Respiratory Diagnostics

1
Respiratory Diagnostics

• Brian J. Payne, DVM
• Carthage Veterinary Service, Ltd.

2
The Presentation

• The actual presentation given at AASV is based on
  actual case studies.
• This PowerPoint presentation (based on PRRS
  diagnostics specifically) describes many of the
  laboratory tests that were utilized for these
  actual cases.

3
One more thing

• It has been an enormous benefit for me in a
  clinical setting to understand each diagnostic
  test that I request so that I can better
  interpret the results.
• I hope this helps!

4
PRRSDiagnostics

• Serology
• PRRSv
• PRRS Viral Antigens
• PRRS Antibodies
• Tissue
• PRRSv
• Other
• Semen PRRSv

5
PRRSSerology

• ELISA
• IFA
• PCR
• RFLP
• Genetic Sequencing
• SVN

6
PRRSIndirect ELISA

• Enzyme-linked immunosorbent assay
• Extremely high sensitivity 100
• 99.5 specificity
• Quick to run
• Indirect method to determine antigen-specific
  PRRSv antibodies

7
Indirect ELISAProcess
8
Indirect ELISAResults

• Bound enzyme allows visual detection
• In-house tests usually () or (-)
• In-lab tests utilize optical densities to give
  continuous range of numbers

University of AZ, 1993
9
Indirect ELISA...Results

• IDEXX HerdChek
• Based on S/P ratio
• sample/positive
• Optical densities (continual range)
• lt 0.3 Negative
• 0.3-0.4 Suspect
• gt 0.3999 Positive

10
Indirect ELISAAntibody Detection

• PRRSv Ab detectable 9-13 days PI
• Peak at 30-50 days
• Rapid decline but still may be detectable in
  gt 10 months (personal experience is gt 2
  years)

11
Indirect ELISALimitations

• Allows evidence of exposure to PRRSv
• However, no information on
• Length of exposure
• Severity of infection
• Which strain is present
• Differentiation of vaccine and wild-type virus
• Variability of immune responses within a given
  population

12
PRRSIFA

• Indirect fluorescent antibody
• Specificity 99.5
• Sensitivity (based on laboratory variation)
• Culture media
• Protocols
• Incubation time
• Cell types
• Technician skill and subjectivity of results
• Lab PRRSv genetic sim. to wild-type PRRSv

13
IFAThe Process
14
IFAThe Results

• Magnitude of Ab titer can be determined unlike
  ELISA

Magar, R., 1993
15
IFAThe Results

• lt 120 or 16 negative
• 120 or 16 or greater positive
• Titer endpoints are subjectively determined,
  therefore variation

16
IFAAntibody Detection

• IgG against PRRSv detectable 7-11 days PI
• Peak at 30-50 days
• Rapid decline but still may be detectable in
  4-6 months

17
PRRSPCR

• Polymerase Chain Reaction
• In vitro amplification of DNA
• RNA of virus extracted from sample
• Reverse transcriptase (RNA to DNA)
• PCR amplifies DNA
• Target most conserved genes
• ORF 6 and ORF 7

18
PCRProcess
Annealing
DNA
Heat Denaturing
Primers and Cooling
Taq Nucleotides
Reverse Transcriptase
30 Cycles
Complementary Strands
RNA
19
PCRThe Process
After 30 cycles, 1000s of replicates
Gel Electrophoresis (Colorimetric, Radioactive,
Fluorometric)
()
(-)
20
PCRAdv / DisAdv

• Rapid turnaround (RT-PCR) - 1-3 days
• High sensitivity and specificity
• Detects Agno need to wait for immune system
  response
• Detection determined by correlation b/w primers
  and PRRSv
• Not indicative of replicating virus

21
PRRSRFLP

• Restriction Fragment Length Polymorphism
• Utilizes RT-PCR of ORF 5 gene
• Envelop proteins
• Prone to mutation
• 3 Restriction Enzymes Used
• Their cutting patterns are assigned numbers
• I.e. 1-4-2, 2-1-2, 2-6-2, 1-5-2

22
RFLPInterpretation

• May differentiate between vaccine virus (2-5-2)
  and wild-type virus
• No indication of virulence
• No indication on cross-protection
• Change in RFLP can be from 1 base pair
  substitutioncareful interpretation
• Little indication of homology (4)

23
RFLPExample
24
RFLPExample
25
PRRS Genetic Sequencing

• Provides exact nucleotide sequence of ORF
• Utilize ORF 5 b/c most variable
• Therefore, more informative than RFLP
• Dendograms (phylogenetic tree) can be made and
  homologies can be seen

26
ORF 5 Genetic Sequencing
ATGTTGGAGAAATGCTTGACCGCGGGCTGTTGCTCGCGATTGCTTTCTTT
GTGG TGTATCGTGCCGTTCTGTTTTGCTGTGCTCGCCAACGCCAGCAAC
AACAGCAGC TCCCATCTACAGCTGATTTACAACTTGACGCTATGTGAGC
TGAATGGCACAGATT GGCTAGCTAACAAATTTGATTGGGCAGTGGAGAG
TTTTGTCATCTTTCCCGTTTT GACTCACATTGTCTCCTATGGTGCCCTC
ACTACCAGCCATTTCCTTGACACAGTC GCTTTAGTCACTGTGTCTACCG
CCGGGTTTGTTCACGGGCGGTATGTCCTAAGT AGCATCTACGCGGTCTG
TGCCCTGGCTGCGTTGACTTGCTTCGTCATTAGGTTT GCAAAGAATTGC
ATGTCCTGGCGCTACGCGTGTACCAGATATACCAACTTTCTT CTGGACA
CTAAGGGCATACTCTATCGTTGGCGGTCGCCTGTCATCATAGAGAA AAG
GGGCAAAGTTGAGGTCGAAGGTCATCTGATCGACCTCAAAAGAGTTGTGC
TTGATGGTTCCGTGGCAACCCCTATAACCAGAGTTTCAGCGGAACAATG
GGGTC GTCCTTAG

gt550 Nucleotides
27
(No Transcript)
28
5.14.06
6.2.06
29
Sequencing and Dendograms

• Phylogenetic analysis
• Nucleotide sequencing

30
(No Transcript)
31
(No Transcript)
32
Genetic Sequencing

• Homologies do not indicate cross-protection
• Homologies do not predict pathogenicity
• Homologies only indicate relatedness

33
PRRSSVN (SN)

• Serum Virus Neutralization
• PRRS SVN not used widely
• Possible direct correlation with protective
  antibodies in vivo
• Alpha and beta procedures
• Beta utilized for PRRS

34
SVNProcedure

• Beta Procedure
• Known levels of standard virus incubated with
  serial dilutions of test serum
• Serum is then added to sensitive cell lines
• Titer of serum Highest dilution of serum that
  neutralizes known dose of virus
• End points are shown by cytopathology

35
SVN Results

• Less sensitive than IFA or ELISA
• Neutralizing Ab produced slow PI ( gt21 days)
• Levels of nAb are pig-dependent
• Detectable 9-28 days PI
• Max titers at 2-3 months PI
• Persistent titers gt 1 year PI

36
SVNInterpretation

• Assumed that shedding of virus is nill if nAb is
  present
• However, PRRS produces carrier-state animals
• REMEMBER
• ARTERIVIRUS

37
PRRSv Detection in Tissues

• Histopathology
• PCR
• VI
• IHC
• DFA

38
PRRSHistopathology

• Interstitial pneumonia
• Thickened alveolar walls
• Pneumocyte hypertrophy/hyperplasia
• Alveolar spaces filled with necrotic debris and
  WBCs

39
PRRSHistopathology
Normal
Interstitial pneumonia
T. Opriessnig, 2004
40
PCRProcess
Annealing
DNA
Heat Denaturing
Primers and Cooling
Taq Nucleotides
Reverse Transcriptase
30 Cycles
Complementary Strands
RNA
41
PRRSVI

• Virus Isolation
• Lung, Tonsil, Lymph nodes
• Autolysis greatly affects sensitivity
• Much easier to find in serum

42
PRRSIHC

• Immunohistochemistry
• Antibody coupled to horseradish peroxidase
• H2O2/Benzidine derivative added
• Colored insoluble precipitate formed

Halbur, AASV
43
PRRSDFA

• Direct Fluorescent Antibody

Fluorescein conjugated to antiviral antibody
PRRSv infected tissue
44
DFAResults

• Quick, Cheap
• Specific
• Less sensitiveautolysis

45
Semen PRRSv PCR
46
Semen PRRSv PCR

• Specific
• Less Sensitive
• Intermittent Shedding
• Detectable Levels gt Infectious Levels

47
Other Respiratory Diagnostics

• I request many of the same diagnostic tests for
  different respiratory diseases.
• If you feel like you are lost when you are out in
  practice, call the lab you are working with and
  the diagnosticians are more than happy to help
  you out and explain more about what they do.